1. Field of Invention
The invention relates to a method for testing the resistance of the HIV-2virus to antiprotease treatment in a patient infected with HIV-2 as well as nucleotide probes usable for such testing.
2. Description of Related Art
Acquired immunodeficiency syndrome (AIDS) is caused by two viruses: HIV-1 and HIV-2. HIV-1 is present throughout the world while HIV-2 is present mainly in western Africa.
Effective antiviral treatments have been in widespread use since 1996 in developed countries where the virus present is HIV-1. Because of their cost, these treatments cannot be used in developing countries where HIV-2 is present.
There are three types of antiretroviral treatments: antiprotease (Indinavir, Ritonavir, Saquinavir, Nelfinavir, and Amprenavir), nucleoside reverse transcriptase (RT) inhibitors (Zidovudine, Didanosine, Zalcitabine, Lamivudine, Stavudine, Abacavir, FTC, and Adefovir), and nonnucleoside RT inhibitors (Nevirapine, Delavirdine, and Efavirenz). These treatments are often given in combination; this is known as multiple-drug therapy.
Antiproteases elicit primary mutations which confer a high degree of resistance but alter the ability of the virus to replicate. Thus, the virus needs to select secondary mutations if it is to be both resistant and able to replicate actively. Also, reverse transcriptase mutations have been described where nucleoside RT inhibitors have been used in combination.
During treatment with HIV-1 infection, particularly if the levels of drug in the bloodstream are inadequate, viral replication is insufficiently inhibited, or rises above the detection threshold of available viral load techniques (the xe2x80x9cviral loadxe2x80x9d measures the quantity of virus genomes in the bloodstream). Because of the high error rate of reverse transcription, mutations take place in the genes targeted by protease and reverse transcriptase treatment. Certain mutations bring about various degrees of resistance to antivirals. Virologic failure occurs in 20 to 40% of patients treated with current multiple drug regimens.
If viruses resistant to one or more substances can be shown for a patient before treatment or if the viral load increases again, the best drug combination for treating HIV-1 can be chosen.
There are currently no published data on mutations in the HIV-2 genome due to the use of antiproteases.
Antiprotease agents that are active against HIV-1 are also active against HIV-2. However, there are no methods available to assist the clinician in determining resistance to antiprotease drugs in patients infected with HIV-2.
The amino acid sequence of the HIV-2 protease is known. In the present application, the numbering system for this amino acid sequence can be deduced from that described in Human Retroviruses and Aids, 1997, Los Alamos National Laboratory, Los Alamos, N.Mex., Chapter II, pp. B10 and B11. The first amino acid in the protease sequence, considered to be position 1 in the present application, is the proline in position 86 of the polyprotein PoL in the ROD strain.
It has now been discovered that antiprotease drugs can bring about mutations in positions 45, 54, 64, 84, and 90 and in positions 10, 46 of the HIV-2 protease and that the mutated viral strains thus appearing are usually resistant to at least one of the antiprotease drugs used.
Hence, the subject of the present invention is a method for testing resistance of an HIV-2 viral strain to antiprotease treatment.
In a preliminary testing phase, this is a method wherein:
a) using known methods, the presence of at least one mutation at one of positions 45, 54, 64, 84, and 90 or one of positions 10, 46 of the protein sequence of the protease of said viral strain in a biological sample taken from a patient infected with HIV-2 is investigated,
b) of the mutations founds in a), those which, after cloning in an HIV-2 virus, do not prevent the virus clone obtained from multiplying in culture in the presence of said antiprotease drug are selected, and
c) if at least one mutation is selected at step b), it is concluded that resistance exists to the antiprotease drug referred to in b).
Of the mutations found in a), those which, when present in a gene cloned in an HIV-2 virus, cause the viral clone not to be significantly prevented from multiplying in the presence of said antiprotease drug are selected. The following procedure may be used to implement step b). The mutations tested are inserted individually into a viral clone by mutagenesis directed by the method described in the article by Kemp et al., J. Virol., 72(6), pp. 5093-5098, 1998. The clones thus obtained are cultured with a xe2x80x9cwild-typexe2x80x9d (i.e. non-mutated) virus clone as a reference in the presence of the various antiprotease drugs able to act against the HIV-2 virus. By measuring the IC50 with a colorimetric test for example (see above Kemp article), the size of the mutation (minor or major) with the various drugs tested can be determined. Thus, one can select the mutations that allow the virus to multiply in the presence of an antiprotease agent as these mutations give rise to strains resistant to this antiprotease.
Of course, in the case outlined in c), the future treatment planned for the patient would be a different antiprotease agent from the agent shown to elicit resistance in this patient.
If step b) did not select a mutation found in step a), it may be concluded that the mutation in question did not elicit resistance by the HIV-2 virus to the antiprotease drug tested for the patient in question.
Obviously, when step b) identifies a mutation that generates resistance to a given antiprotease drug, step b) of the method described above need not be carried out in the future. In this case, step a) would suffice because the link between the mutation and resistance to the antiprotease drug would be established once and for all; one can go on directly to step c) and conclude that there is resistance to the antiprotease agent studied.
The invention relates in particular to a method for detecting any resistance of an HIV-2 viral strain to treatment by an antiprotease drug in which the presence of at least one mutation chosen from the following mutations:
K45R, I54M, I64V, I84L and L90M, or V10I, I46V, and I82M,
in the protein sequence of the protease of said viral strain is investigated and in which said resistance is concluded to exist if said mutation or said mutations is or are present.
The conventional notation in the present application for describing a mutation is as follows: The number indicates the position in the amino acid sequence of the HIV-2 protease. The letter to the left of the number is the amino acid of the wild-type strain in the international classification, with the one-letter code. The letter to the right of the number is the amino acid, in the same classification, resulting from a mutation.
xe2x80x9cWild-type strainxe2x80x9d is understood to be a viral strain that has not mutated after treatment with an antiprotease.
To identify a mutation in the protein sequence of the protease of the viral strain in question, it is preferable to look for a corresponding mutation in the nucleotide sequence of the gene of said protease. These mutations can be tested on the DNA or the RNA. Of course, in looking for a mutation in the protein sequence by seeking a mutation in the nucleotide sequence, degeneration of the genetic code would be taken into account, namely a given amino acid can be coded by different codons. This mutation assay can be done in the nucleotide sequence by known methods, particularly by hybridization or sequencing techniques.